There is an ever increasing demand for inexpensive and environmentally acceptable viscosifiers, bioemulsifiers and biodegradable polymers. Exopolysaccharides are an example of compounds that are useful for these purposes because of their distinctive rheological properties. Exopolysaccharides, such as for example gellan, welan and rhamsan, are produced commercially for applications in foods, cosmetics, and in oil-field production, and for other applications. Each exopolysaccharide displays a different characteristic set of aqueous rheological properties including resistance to shear, compatibility with various ionic compounds, and stability to extreme temperatures, pH and salt concentrations.
Exopolysaccharides can be produced through bacterial fermentations. Different strains of the genus Sphingomonas produce exopolysaccharides including gellan, welan, rhamsan, S-88, S-7, S-198, NW11, and S-657, to name some examples (Pollock 1993, J. Gen. Microbiol. 139:1939-1945). There are many other exopolysaccharides made by other strains of Sphingomonas bacteria. The exopolysaccharides produced by Shpingomonas bacteria are referred to as “sphingans” with reference to the common genus as a source. At least three sphingans (gellan, welan, and rhamsan) are produced commercially by large scale submerged fermentation.
The biotechnology industry has responded to the demand for exopolysaccharide compounds by increasing the availability of a variety of bacterial exopolysaccharide products that are acceptable for commercial use. Although many of the bacterial exopolysaccharide products offer a wide range of attractive improvements over synthetically produced materials, they remain relatively expensive to produce. The expense is generally associated with costs of recovery and purification of the desired product.
Higher fermentation yields of exopolysaccharides have occurred as a result of improvements and alterations of bacterial strains, and better understanding of bacterial biosynthesis and optimization of fermentation conditions. This satisfies one of the important steps in recovering adequate amounts of the polymer for potential industrial applications. However, increased exopolysaccharide concentration in the fermentation process results in increased viscosities which require higher inputs of energy to effectively disperse oxygen and nutrients in the fermentation broth. Hence, fermentations that provide higher exopolysaccharide yields have resulted in correspondingly higher production costs.
Recovery of exopolysaccharides remains a difficult and costly step. Bacterial strains from the genus Shpingomonas produce exopolysaccharides which remain attached to the cell surface (Pollock et al. 1999, J. Indust. Microciol. Biotechnol. 23: 436-441). The attached polymers form a capsule around the bacteria. The capsule of polysaccharide is not readily separated from the bacteria. Even after diluting a fermentation broth with sufficient water to reduce the viscosity, the capsule remains attached to the bacterial cells and the cells cannot be separated from the capsule by centrifugal sedimentation. Other physical or chemical methods are required to separate the cells from the capsule. For example, partial hydrolysis of the polysaccharides with acid can be used to release most of the polysaccharide from the cells by randomly breaking the polymer chains near to the point of attachment to the cell.
Recovery of exopolysaccharide, regardless of the conditions used to produce it, typically involves a precipitation step. The precipitated exopolysaccharide is then recovered by centrifugation. A typical method for recovering gellan and welan gums is a follows. Immediately after fermentation the culture broths are heated to at least 90° C. to kill the living bacteria. Both gums are then separated from the culture broth by precipitation with approximately 2 volumes of isopropylalcohol, and the precipitated polysaccharide fibers are collected, pressed, dried and milled. The alcohol is recovered by distillation. In this most simple process the polysaccharide remains attached to the cells, such that when the dried and milled polysaccharides is resuspended in water the solution is not transparent. In the case of gellan gum additional steps can be introduced to purify the polysaccharide away from the bacterial cells so that the resuspended product is more transparent. Before the alcohol precipitation, the culture broth is centrifuged or filtered or both while the temperature is maintained above the critical transition temperature between a highly viscous state and a liquefied state which is amenable to centrifugation or filtration. These processes are disclosed in U.S. Pat. No. 4,326,052 (gellan); U.S. Pat. No. 4,326,053 (gellan); U.S. Pat. No. 4,342,866 (welan); U.S. Pat. No. 3,960,832 (S-7); and U.S. Pat. No. 4,535,153 (S-88), which are hereby incorporated by reference.
A major inefficiency associated with a typical product recovery protocol is incomplete recovery of the exopolysaccharide. Bacterial exopolysaccharides are attached to the producing cells with varying degrees of tenacity. Those bacteria that have relatively securely attached exopolysaccharides are less likely to shed them into the medium, thus reducing the amount of exopolysaccharide available for recovery in the precipitation step and increasing the process steps needed to separate the exopolysaccharides from the producing cells.
Advantages, features and characteristics of the present invention will become more apparent upon consideration of the following description and the appended claims.